Does Lactic Fermentation Influence Soy Yogurt Protein Digestibility A Comperative Study Between Soymilk and Soy Yoghurt at Different PH
Does Lactic Fermentation Influence Soy Yogurt Protein Digestibility A Comperative Study Between Soymilk and Soy Yoghurt at Different PH
Does Lactic Fermentation Influence Soy Yogurt Protein Digestibility A Comperative Study Between Soymilk and Soy Yoghurt at Different PH
Xin Rui, Qiuqin Zhang, Jin Huang, Wei Li, Xiaohong Chen, Mei Jiang, Mingsheng
Dong*
Province, P R China
Mingsheng Dong
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jsfa.9256
digestibility when ingested. In the current study, soymilk (pH 6.3) and soy yogurt (SY)
at four varied pH (6.0, 5.7, 5.4, 5.1) were subjected to in vitro gastrointestinal
in 330.0 min. Decline of pH made D[4,3] and D[v,90] increased from respectively
0.81 to 97 μm and 1.82 to 273 μm. Predominant proteins lost their solubility between
SY-5.1 to show particles with predominant peak at around 10 μm and lower soluble
Cleavage pattern of soy protein during GIS was barely affected by sample pH. But
less quantity of band at 33.9 kDa was found in SY-5.7, SY-5.4 and SY-5.1.
digestibility. With the process of protein coagulation, SY-5.7, 5.4, and 5.1 had lower
Proteins from plant sources are received increasing notice due to many
soybeans after removal of insoluble residuals (okara).1 Aside from its benefits on
soy protein and made it a set-type yogurt like product.4,5 LAB during their growth
slowly release protons and allowed decline of pH which made soymilk proteins
proteins are behaved particulate in nature other than network gels.7 This has made soy
protein gels varied texture and microstructure compared to that of casein gels.8 As
many studies have focused on LAB induced gelation process,6,9 fewer studies have
Turgeon and his group in 2011 pointed out macronutrient like proteins needs to
be released from the food matrix to obtain real benefit of this compound.10 The
procedure of releasing nutrients from a complex food system to the intestinal lumen
objective of this study is to find data that indicates that coagulation of soy protein
indicator during growth of LAB and also determines process of protein coagulation.
In the current study, soy yogurt was prepared at different pH levels and was
study was conducted in terms of particle size distribution, soluble protein content, and
electrophoretic profiles.
2.1. Materials
China and were stored at 4 °C until use. Molecular mass standard (20-150 kDa) was
saliva, A1031), pepsin (from porcine gastric mucosa, P7125), pancreatin (from
porcine pancreas, P7545), and bile salts (from porcine, B8631) were obtained from
grade.
Lactobacillus plantarum B1-6 was isolated in our lab from Krigiz boza, which is
a traditional type of fermented cereal based drink from Xinjiang province of China.
The LAB culture were propagated twice in de Man-Rogosa and Sharp broth
(MRS, pH 6.2) at 37 °C for 24 h and they were subsequently inoculated into MRS
broth and cultured for another 16 h. The cells were and washed twice with sterile
Soybeans were selected, rinsed and soaked in distilled water for approximately 12
h and then drained and homogenized in 9-fold of distilled water to make slurry.
Filtration was then carried out using a 200-mesh screen cloth to remove the insoluble
soybean residue (okara). The filtrate, soymilk, was then heated at 108 oC for 15 min to
reduce the endogenous microbiota in the raw material. Part of the prepared soymilk
was left to cool down and inoculated with L. plantarum B1-6 at inoculation ratio of
3% (v/v) to make soy yogurt (SY). Fermentation temperature was set at 37 oC. An
analysis.
A Physica MCR301 rheometer (Anton Paar, Austria) was used for the rheological
was loaded between parallel plates (50 mm of diameter) with gap set at 1.0 mm. Edge
of samples was covered with silicon oil to prevent evaporation. Gelation process was
37 °C. Storage modulus (G2) and loss modulus (G22) were recorded every 2 min for 6 h.
Gelation time was defined at the point when G2e 1 Pa (log G2= 0).16 The corresponded
pH value was defined as gelation pH. According to the gelation pH, fermentation was
terminated at different times when pH of the culture dropped to 6.0, 5.7, 5.4 and 5.1
and samples collected were named respectively SY-6.0, SY-5.7, SY-5.4, and SY-5.1.
These samples, together with sample of soymilk were subjected to the following
study.
counts were expressed as log cfu ml-1. Three replicates were conducted.
diameter×2.0 cm height) from the central part with a stainless steel cutter. A 5 kg load
cell was used and the test speed of 5.0 mm/s. The sample was compressed by a 5 cm
diameter cylinder probe to 35% deformation. The pre-test compression speed of the
probe was set as 1.0 mm/s. Hardness, springiness, cohesiveness, and gumminess were
Artificial saliva, gastric and duodenal juices were prepared to simulate human
The ratio of food to digestive juices was set to be 2.5 (food) to 1.0 (saliva) to 1.5
(gastric juice) to 1.0 (bile) to 1.0 (pancreatic juice) based on volume units to mimic
±-amylase (0.2 mg/mL, w/v) in phosphate buffer (20 mM, pH 7.0). Artificial gastric
pancreatic juice and bile acids were prepared by dissolving pancreatin (0.4 mg/mL,
w/v) and bile salts (0.4 mg/mL, w/v) respectively in phosphate buffer (10 mM, pH
process and then 10 mL artificial saliva was added. The mixture was placed in a
shaking water bath (SWB series, Biobase, Shandong, China) at 55 rpm, 37 oC for 3
min for simulating of buccal digestion. Subsequently, the slurry was adjusted to pH
2.0 and artificial gastric juice (15 mL) was added. The mimic gastric digestion was
artificial bile acids (10 mL) and pancreatic juice (10 mL) were added to simulate
Aliquots were taken before digestion (P0), at the end of buccal digestion (P1), gastric
digestion (P2), and duodenal digestion (P3), respectively. All collected samples were
boiled for 5 min to terminate the enzymatic hydrolysis. Samples for particle size
distribution determination were taken immediately for the analyses, whereas samples
MA, USA). The refractive index of the scatterers of the sample solution was set as
1.46 and a refractive index of the water dispersant was set as 1.333. Samples were
dropped into the chamber filled with water until an optimum dilution was reached.
The volume-weighted mean diameter D[4,3] was calculated to represent the size of
gel particles, whereas D[v,0.90] was also given to describe the diameter below which
90% of the volume of particles are found. The analysis was conducted in triplicates.
Total soluble protein content was determined according to the Bradford assay
using a previous protocol with bovine serum albumin as a standard.21 Triplicates were
2.10. Electrophoresis
(Bio-Rad Laboratories, Inc., Hercules, CA, USA) with voltage 60 V for stacking gel,
and followed by 120 V for separating gel. A pre-stained molecular mass standard
(20-150 kDa, Sangon Biotech Co., Shanghai, China) was used. Gels were scanned
with Image Scanner III (GE Healthcare Biosciences, Uppsala, Sweden) and then
analyzed by using Quantity One software, version 4.6.2 (Bio-Rad Laboratories, Inc.,
used to determine the significant differences between means (P<0.05) using IBM
allowed gradually decline of pH and increments in the storage modulus (G2) and loss
modulus (G22) during 6 h of fermentation (Fig 1A). G2 and G22 remained unchanged
during 0-140 min of fermentation and then increased rapidly from 140 min to 200 min.
Gelation time, which was assigned when the value of G2 was higher than 1 Pa (log
G2=0) based on the previous literature,16 was 163±6 min. The corresponded pH was
which were 6.0, 5.7, 5.4, and 5.1, to evaluate coagulation of soy protein during
SY-6.0, SY-5.7, SY-5.4, and SY-5.1 hereafter. It took 141.0±3.2 min, 204.0±1.2,
257.0±2.5 min and 330.0±4.1 min to form samples SY-6.0, SY-5.7, SY-5.4, and
lactic bacteria cell counts is shown in Fig. 1B. The viable bacteria count of L.
log cfu/ml in SY-6.0, SY-5.7, SY-5.4, and SY-5.1, respectively, indicating a steady
gumminess were evaluated on soy yogurts collected at different terminal pH (Table 1).
SY-6.0 and SY-5.7 were either remained at emulsion state or had very weak texture
which were failed in deformation process, thus the results were not shown in the table.
respectively. Springiness and cohesiveness values between the two gels appeared to
related to a more compact structure. This was in agreement with previous results.22
Particle size distribution was followed for soymilk and SY samples during in
vitro gastrointestinal digestion simulation (GIS) (Fig. 2A-C). Soymilk, after buccal
digestion, showed a bimodel profile with size of particles peaked at approximately 0.5
μm and 2 μm (Fig. 2A). This was in agreement with results observed in a previous
study.6 The authors suggested the smaller particle population represented soymilk
protein particles whereas the larger ones were mostly related to fat globules.
respectively 0.81 to 97 μm and 1.82 to 273 μm (Fig. 2A). In sample SY-6.0, 0.1-1 μm
particle population was reduced and a new peak at approximately 6 μm has emerged,
modified in SY samples obtained at pH 5.7 or lower. SY-5.7, 5.4 and 5.1 gave
monodisperse distribution with particles peaked at around 12 μm, 20 μm, and 23 μm.
This has suggested a uniform structure has formed after mimic mastication for those
samples (Fig. 2A). Evolvement of larger size particles upon mastication in samples
with lower pH probably related to higher gel stiffness and more compact structure
Gastric digestion has an significant impact on particle size distribution for all
investigated samples (Fig. 2B). D[4,3] and D[v,90] of SY-5.4 and SY-5.1 were
and D[v,90] were obtained for soymilk and SY-6.0 digesta which suggestive of
showed D[4,3] ranged from 21 to 27 μm and had very similar bimodal distribution
smaller (0.01-1 μm) and larger (100-500 μm) particles, implied an extensive
destabilization of the emulsion. In this stage, soymilk digesta possessed the largest
higher quantity of larger sizes particles (>100 μm) observed in soymilk digesta.
interesting soymilk digesta formed higher quantity of large aggregates than all SY
mainly stabilized by van der Waals attraction, hydrogen bonding and hydrophobic
was found at lower particle range (0.01-1 μm). SY-6.0 and SY-5.7 showed a broad
peak at lower particle sizes (0.01-1 μm) which was similar with soymilk digesta.
However, this peak was absent in sample SY-5.4 and SY-5.1. In addition, sample pH
also decided height of the peak at around 10 μm. The peak height, corresponding to
the particles were varied upon different sample pH, although all samples represented
similar profiles in previous gastric stage. It appeared matrix formed at gastric stage
was maintained higher integrity in this stage for SY samples especially for those with
lower pH (SY-5.4 and 5.1). Size of the particle may further influence susceptibility of
phases are shown in Fig. 3. At P0 stage, soymilk presented the highest soluble protein
content (5.2 mg/mL) followed by SY-6.0 (3.9 mg/mL), whereas drastically lower
values (0.3-0.4 mg/mL) were observed in SY-5.7, 5.4, and 5.1 with no significant
differences between them (P>0.05). It seems most soymilk proteins loss their
bacteria has a similar gelation mode to that of glucono-´ -lactone but showed a higher
gelation pH at 6.29.6 During the first phase of GIS digestion, mimic mastication (P1)
resulted in a similar result with that of P0 in all investigated samples expect SY-6.0,
which suggested mechanical chewing and incorporation of ±-amylase had little effect
on soluble protein content of SY. SY-6.0 gave a higher result at P1 stages and the
maintained higher soluble protein contents than that of SY-5.7, 5.4 and 5.1,
differences between them were dramatically reduced and the latter three digesta
afforded climbing values. This was in accordance with the results found in particle
different samples. In the subsequent duodenal (P3) stage, soymilk and SY-6.0 digesta
showed the highest soluble protein contents and lower soluble protein contents were
obtained in SY-5.7, 5.4, and 5.1 in descending order. SY-5.7, 5.4, and 5.1 showed
SY-6.0 showed similar results (1% reduction) with soymilk digesta and no significant
corresponding to greater protein aggregates which might be too large to cross gut
5.4, and 5.1 in descending order. For SY samples, especially SY-5.7, 5.4, and 5.1,
the sample pH related to more a profound effect. With the decrement of sample pH,
less proteins were “released” during in vitro GIS to become soluble which might
of cow milk has a different manner when subject to gastrointestinal digestion. The
study showed yogurt possessed a higher soluble protein content than that of cow milk
after intestinal phase of GIS.15 This might be due to different gelation behavior of soy
3.5 Electrophoresis
condition (Fig. 4). Soymilk collected at P0 stage afforded several bands with MM
(molecular mass) ranged between 20-100 kDa. The predominant six bands, showed
with the number of 1-6, possessed molecular mass of 85.2 kDa, 76.6 kDa, 48.6 kDa,
40.0 kDa, 35.1 kDa and 20.1 kDa, respectively in an ascending order of the band
globulin ± subunit (band 2), and 7S globulin ² subunit (band 3), 11S A3 subunit (band
terminal pH, all SY samples showed protein composition including five major bands
(7-11) with corresponding MM of 61.5 kDa, 55.4 kDa, 48.9 kDa, 40.0 kDa, and 25.7
kDa. It appeared 7S globulin ±� subunit, ± subunit and 11S acidic subunits were
respectively bands 3 and 4 but both were in presences of reduced contents. The absent
proteins might be distributed in the corresponding precipitates thus were not appeared
in the supernatant fraction. It can be presumed that lactic fermentation has triggered
different protein subunits into insoluble fraction at varied pH. A previous study
proteins including 7S subunits and 11S subunits and made them disappeared from the
All SY samples except SY-6.0 showed similar electrophoretic profile before and
after buccal digestion (P1). Several additional proteins were seen on lane P1 (SY-6.0)
possessed identical MMs with band 1, 2, and 5. This has indicated 7S globulin ±�
subunit, 7S globulin ± subunit, and 11S acidic subunits were probably migrated to
This phenomenon was not seen in the SY-5.7, 5.4 and 5.1 (Fig. 4). No additional
bands were observed in the latter three samples but bands 7-11 were shown with
higher intensity. The results indicated SY samples starting from SY-5.7 presented
higher interacting forces between soymilk proteins thus hindered releasing of those
protein into soluble fraction. These results were in accordance with previous findings
fractions. Bands 1-6 were disappeared and seemed to be degraded to result in a few
degradation products, namely, bands 12-14 with identified MM of 33.9 kDa, 26.4
kDa, and 22.7 kDa. SY samples at P2 stage showed similar electrophoretic profile
with soymilk in terms of MMs of degradation products (as pointed by arrows). In the
subsequent duodenal digestion (P3) soymilk and SY-6.0 digesta showed similar
profile but with higher intensity in bands 12-14, indicating accumulation of these
products. For sample SY-5.7, 5.4 and 5.1, higher intensity was found in bands 13 and
14, whereas band 12 was observed with reduced intensity. The results indicated
sample pH has little effect on the MMs of cleavage products released during
protein coagulation process may not affect the cleavage pattern of soy protein during
in vitro GIS. But quantity of the products (bands 12-14) might be affected.
4. Conclusion
pH of 5.86±0.02. Soy yogurt (SY) was collected at four varied pH, namely, 6.0, 5.7,
5.4 and 5.1, were subjected to in vitro gastrointestinal digestion and their protein
digestibility was compared. The results suggested matrix of soy yogurts was greatly
interrupted by gastric (P2) and duodenal digestion (P3) and modifications in particle
size distribution, soluble protein content and electrophoretic patterns were observed.
At the end of duodenal digestion, SY samples were shown lower quantity in 100-500
lower soluble protein content (SY-5.7, 5.4, 5.1), and fainter bands at 33.9 kDa which
probable a digestive product (SY-5.7, 5.4, 5.1) compared to that of soymilk digesta.
SY-6.0, after buccal digestion (P1), showed similar soluble protein content and
allowed the formation of a more compact structure and larger aggregates might
the final product will probably be an important index for maintain protein quality of
Acknowledgements:
the Fundamental Research Funds for the Central Universities (KJQN201647 &
KYZ201745). The author would also like to acknowledge the funding from the
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LIST OF FIGURES
Fig. 1 Microbial and acid gelation behavior of soymilk during the growth of L.
plantarum B1-6. (A) Changes in pH (△), Storage modulus G’ (■), and Loss
modulus G’’ (○); (B) Changes in viable lactic acid bacteria cell counts.
Fig. 2 Particle size distribution of soymilk and SY samples collected at varied phases
of in vitro GIS (A) P1: after simulated buccal digestion, (B) P2: after simulated
represented the soymilk (blue closed circle), SY-6.0 (red closed square), SY-5.7
(green closed triangle), SY-5.4 (purple cross), SY-5.1 (blue open triangle). Data are
expressed as mean ± SD from triplicate experiments. Different letters within the same
Fig.3 Soluble protein contents of soymilk and SY samples at varied phases of in vitro
GIS (A) P0-before the GIS; P1-after simulated buccal digestion; P2-after simulated
gastric digestion; P3- after simulated duodenal digestion. The error bars represent the
buccal digestion (P1); P2-after simulated gastric digestion (P2); P3-after simulated
duodenal digestion (P3). 1-14 represented predominant bands observed before and
Fig. 1
A D[4,3] D[v,0.90]
(μm) (μm)
Soymilk 0.81±0.02d 1.82±0.03c
SY 6.0 1.57±0.04d 5.00±0.10c
SY 5.7 20±3c 34±6c
SY 5.4 50±11b 119±36b
SY 5.1 97±29a 273±92a
B D[4,3] D[v,0.90]
(μm) (μm)
Soymilk 27±1a 58±8a
SY 6.0 24±1abc 44±6ab
SY 5.7 25±2ab 25±1c
SY 5.4 22±1bc 23±3c
SY 5.1 21±2c 32±13bc
C D[4,3] D[v,0.90]
(μm) (μm)
Soymilk 43±1a 164±3a
SY 6.0 29±2c 95±12b
SY 5.7 18±1e 23±1d
SY 5.4 33±1b 96±10b
SY 5.1 24±1d 38±2c